1. Field of the Invention
The present invention relates generally to prothrombin time blood coagulation testing, and more particularly to dry reagent prothrombin time test articles; and prothrombin time test methods which rely upon the rehydration of dried thromboplastin in the presence of blood or plasma.
Blood coagulation tests may be performed for a variety of purposes, including determination of the bleeding susceptibility of patients undergoing surgery and monitoring of patients undergoing anti-coagulation therapy for the prevention of blood clots. A variety of coagulation tests are presently in use. One of the most popular is the "prothrombin time" (PT) test which relies on induction of the extrinsic coagulation pathway by activation of coagulation protease factor VII by thromboplastin in a blood sample to be tested.
The extrinsic coagulation pathway results in the production of thrombin, which is a proteolytic enzyme which catalyzes the conversion of fibrinogen to fibrin. Such conversion is an essential function in the clotting mechanism.
Heretofore, such blood coagulation tests have tended to be complex, with performance generally limited to clinical laboratories. While such centralized testing may be adequate for surgical patients, visiting a doctor's office or a clinic on a regular basis to monitor anti-coagulation therapy is less acceptable. Thus the need for a convenient and practical home coagulation monitoring test is apparent.
Successful home blood tests have been devised for other chemistries, such as cholesterol and glucose. Among the most suitable devices for home use are dry reagent test devices containing all test components pre-mixed, and preserved in a dry form suitable for long term storage. These dry reagent test devices include test strips, small plastic chambers, and the like.
Dry reagent assays typically have lower precision than liquid phase assays. This is because of structural differences between the two. Liquid phase assays have a very simple and uniform reaction zone which is formed by the walls of the vessel in which the reaction takes place. By contrast, dry reagent assays have a complex reaction zone, formed by the surfaces of the dry reaction chemicals and the support matrix. Typically, dry reagent support matrices are porous membranes or capillary gap chambers, which have higher surface to volume ratios than the reaction cuvettes typically used for liquid phase assays. Thus, test samples entering a dry reagent assay have a much greater chance of interacting with the assay components in a variable manner, resulting in a loss of precision. If the precision of the dry reagent assay is too low, the test can be rendered essentially useless for practical clinical applications.
A test's precision can best be described by the coefficient of variance, which is the ratio between the scatter in test results obtained in testing large numbers of identical samples, divided by the magnitude of the overall test signal. If the scatter in test replicates is large relative to the overall test signal, the test has a poor precision. Conversely, if the scatter in test results is small relative to the overall test signal, the test has a better precision. Thus, there are two ways to improve the precision of a test. The first is to reduce the scatter in test results while maintaining the signal magnitude, and the second is to increase the signal magnitude while keeping the scatter essentially constant. Often, the second method, increasing the magnitude of the test signal, is the most practical way to improve precision.
Blood and plasma contain a family of serine proteases that regulate the clotting process. These serine proteases are called coagulation "factors", are typically designated with Roman numerals (with an "a" suffix added if the factor is in an enzymatically active state), and operate in a precisely regulated amplification cascade to form blood clots at the site where tissue has been injured. These blood clots act to stop bleeding at the injury site. This particular mode of blood coagulation is termed the "extrinsic" coagulation pathway.
Normal tissue contains a membrane bound glycoprotein, called tissue factor, which is liberated when the tissue is injured. The extrinsic coagulation process begins when this tissue factor forms a complex with coagulation factor VII and/or VII(a). This tissue factor--factor VII(a) complex in turn activates factor X, which in concert with co-factor V, transforms the inactive prothrombin protease into the active thrombin enzyme. Thrombin then transforms fibrinogen into fibrin, which forms the actual blood clot. This process is described in detail in Tissue Factor and Hemostasis, Y. Nemerson, Blood 71 (1), 1-8, 1988, the full disclosure of which is incorporated herein by reference. The nomenclature in this document follows that of Nemerson. The prothrombin time test stimulates this coagulation pathway in vitro, using a tissue extract called thromboplastin to initiate the coagulation pathway.
Conventional PT assays have usually employed thromboplastin purified from an aqueous extract of acetone dried brain tissue. This crude extract contains many components. The active component of normal thromboplastin is a poorly defined mixture of factor VII reactive molecular complexes, each formed by an interaction between "tissue factor" proteins, and the mixture of brain lipids remaining after extraction. By contrast, synthetic recombinant thromboplastin (r-DNA thromboplastin) consists of a relatively simple, well defined complex formed by purified recombinant tissue factor protein, and a purified artificial lipid population.
It is known that, in liquid phase assays, different thromboplastin preparations can improve or reduce discrimination between blood samples having different prothrombin times. Those thromboplastins with a greater discrimination are termed "more sensitive." The liquid phase sensitivity of a thromboplastin preparation is graded by use of the international sensitivity index, or ISI. This ISI value is found by plotting, on a logarithmic scale, the prothrombin time seen with a thromboplastin lot in question, versus the prothrombin time values seen with a standardized lot of thromboplastin (normally defined to have an ISI value of 1). The ISI value is the slope of the resulting line, multiplied by the ISI of the reference thromboplastin.
The scale itself is somewhat non-intuitive. More sensitive thromboplastins have lower ISI numbers, typically around 1.0, and less sensitive thromboplastin lots have higher ISI numbers, typically around 2-3. The molecular explanation for the difference between lots is not totally understood at this time.
In the case of prothrombin time assays, high precision is of extreme clinical importance. Depending upon the severity of scatter, a blood sample with an actual International Normalized Ratio (INR) of 2.5 might, with an imprecise test, be reported as having an INR of 2.0 or 3.0. Such imprecision can lead to different clinical decisions. A physician might decide to increase anticoagulant dosage if the INR 2.0 result were obtained, and decrease anticoagulant dosage if an INR 3.0 result were obtained. Both decisions would have a significant impact on the patient well being, and both might be erroneous, since a "correct" test result of INR 2.5 might result in the decision not to adjust anticoagulant dosage at all.
Because of these clinical issues, methods to improve the precision of a dry reagent prothrombin time assays are thus of considerable importance to the field.
2. Description of the Background Art
Thromboplastins and tissue factors produced by recombinant DNA technology are described in U.S. Pat. Nos. 5,110,730 and 4,966,852. Methods to prepare thromboplastins with higher sensitivity for liquid phase assays are described in U.S. Pat. Nos. 5,270,451 and 4,416,812. Use of recombinant tissue factor in prothrombin time tests is described in Recombinant Tissue Factor as Substitute for Conventional Thromboplastin in the Prothrombin Time Test, A. Tripodi, A. Arbini, V. Chantarangkul, and P. Mannucci, Thrombosis and Haemostasis 67(1) 42-45 (1992). Use of the International Normalized Ratio (INR) for accounting for differences in sensitivity between thromboplastin types is described in Calibration of Reference Thromboplastins and Standardization of the Prothrombin Time Ratio, T. Kirkwood, Thromb. Haemostasis 49 (3) 238-244 (1983), and in Requirements for Thromboplastins and Plasma used to Control Oral Anticoagulant Therapy, WHO Expert Committee on Biological Standardization, 33rd Report, WHO Tech Rep. Ser 1983; 687:81-105. The mechanism by which tissue factor activates factor VIIa in the prothrombin time clotting cascade is discussed in The Interaction of Human Factor VIIa with Tissue Factor, S. Kirshnaswamy, The Journal of Biological Chemistry 267 (33) 23696-23706 (1992), the disclosures of which is incorporated herein by reference.
Dry reagent prothrombin time tests using test strips with dried thromboplastin are described in copending application Ser. No. 08/196,816 and U.S. Pat. No. 5,344,754, the full disclosures of which are incorporated herein by reference.